Summary Over 40% of HIV-positive individuals in the United States engage in substance use. This not only represents a major cause of enhanced morbidity and mortality, but also is associated with increased risks of neurocognitive disorders, such as HAND. Neurons possess refined systems for maintaining constant communication with glia through propagation of ?Off? and ?On? signals controlling microglial activation states. Using HIV latency models in immortalized human microglial cells (hglia/HIV), we have shown previously that cellular activation and inflammatory responses induce HIV production. Remarkably, co-culture of productively infected microglia with an excess of healthy neurons leads to viral silencing. We have also shown that hglia/HIV cells can migrate into brain organoids where they become silenced. However, damaging neurons with a variety of agents, including methamphetamine (METH), a frequently-used abuse substance among HIV-infected individuals, produce reactivation signals for HIV, and this initiates a cycle of microglial activation and further neuronal damage. This cycle of shutdown and reactivation seems to parallel the M1 to M2 transition model of microglial cells, much as HIV latency in T cells is a product of the natural transition of effector cells to resting memory cells. In this proposal, we seek to define the key signals mediating the cycle of viral silencing and reactivation in microglial cells by neurons in the context of iPSC-derived cerebral organoids. This multidisciplinary investigation is designed as a close collaboration between the laboratories of Dr. Jonathan Karn (CWRU, HIV molecular biology), Dr. Anthony Wynshaw-Boris (CWRU, iPSC cells, brain organoids), Dr. Kurt Hauser (VCU, neurobiology and drug abuse), and Dr. Pamela Knapp (VCU, brain organoids). To avoid the limitations of working with transformed cells, we have recently initiated experiments using co-cultures between iPSC-derived cerebral organoids and microglia. Using co-cultures between iPSC-derived cerebral organoids and microglia, we will thoroughly test the hypothesis that the exaggerated responses of HIV-infected microglia to neuronal damage leads to enhanced neurodegeneration. We will also test the hypothesis that exposure to METH, given this background of faulty microglia-neuron crosstalk, enhances HIV replication. Using genome editing approaches, we will identify the specific contribution of ?On? and ?Off? receptor systems in controlling HIV latency in microglia, and study how METH impacts neuronal-microglial signaling to augment HIV production. In parallel with our genetic investigations, we will also evaluate a number of pharmacological agents against microglial receptors, HIV transcription inhibitors, and mediators of inflammation in order to define therapeutic approaches that might be expected to slow the development of HAND, especially in patients who abuse drugs.